Data Analysis from Michaelis-Menten Kinetics: Ins and Outs

Keleti, Tamás

doi:10.1007/978-1-4613-3255-8_21

Cited by 3 publications

(1 citation statement)

References 98 publications

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“…Whatever makes the progress curves deviate from what would be generated using both models is not clear. Plausible hypotheses which may explain these deviations are the presence of isoenzymes, association‐dissociation of oligomers, interaction of the enzyme with other proteins, macromolecules or membranes and inactivation [41].…”

Section: Resultsmentioning

confidence: 99%

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae

Martins

Mendes

Cordeiro

et al. 2001

European Journal of Biochemistry

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The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis.Initial rate analysis was performed by hyperbolic regression of initial rates using the program hyperfit. Glyoxalase I exhibited saturation kinetics on 0.05±2.5 mm hemithioacetal concentration range, with kinetic parameters K m 0.53^0.07 mm and V (3.18^0.16) Â 10 22 mm´min 21 . Glyoxalase II also showed saturation kinetics in the S-d-lactoylglutathione concentration range of 0.15±3 mm and K m 0.32^0.13 mm and V (1.03^0.10) Â 10 23 mm´min 21 were obtained.The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program gepasi. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis±Menten model. The kinetic parameters obtained, K m 0.62^0.18 mm and V (2.86^0.01) Â 10 22 mm´min 21 , were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis±Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with K m . The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.

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Section: Resultsmentioning

confidence: 99%

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae

Martins

Mendes

Cordeiro

et al. 2001

European Journal of Biochemistry

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Coupled Reactions and Channelling: their Role in the Control of Metabolism

Keleti

1990

Control of Metabolic Processes

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Two rules of enzyme kinetics for reversible Michaelis‐Menten mechanisms

Keleti

1986

FEBS Letters

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In a Michaelis-Menten type reversible enzyme reaction (one substrate, one product) the rapid equilibrium kinetics in one direction excludes rapid equilibrium in the reverse direction. If rapid equilibrium functions in any direction, in the reverse reaction van Slyke type 'kinetic constant' appears in the rate equation independently of whether steady state is reached in finite time or the final equilibrium is attained at t = m. If the reaction proceeds in one direction with rapid equilibrium and in the reverse direction with steady-state kinetics, the thermodynamic equilibrium of the reaction determines that a higher equilibrium concentration of product (or substrate) can be reached only with steady-state kinetics.Let us consider a reversible Michaelis-Menten type mechanism [1,2], where a single product is formed through a single enzyme-substrate complex from a single substrate (scheme 1). E+S -where E stands for enzyme, S for substrate and P for product. The kl and k-2 are second order, k-t and kz first order rate constants. In this case, considering the initial velocity conditions in both directions:where KM,S and KM,p are the Michaelis constants of the substrate and product, respectively. If k2 < k_, in the forward reaction, the rapid equilibrium kinetics holds. Consequently in this case

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Data Analysis from Michaelis-Menten Kinetics: Ins and Outs

Cited by 3 publications

References 98 publications

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae

Coupled Reactions and Channelling: their Role in the Control of Metabolism

Two rules of enzyme kinetics for reversible Michaelis‐Menten mechanisms

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